We are examining structure-function relationships for cytochrome P450, with particular emphasis on the interactions between P450 and other mixed function oxygenase proteins and binding of substrates and ligands to P450. Previous studies on the interactions of P450 with NADPH cytochrome P450 reductase and cytochrome b5 were extended to examine the specific amino acid residues involved in these protein-protein interactions. Molecular modeling and multiple sequence alignments were used to predict intermolecular interface regions, and peptides corresponding to these regions were synthesized by the solid phase technique. Binding of cytochrome b5 to rat P450 2B1 was inhibited (by 75%) by a peptide corresponding to P450 residues 116-134. Substitution of Glu for Lys-122 nearly abolished this inhibition. These results indicate a role for this P450 region as well as a specific basic residue in the P450-cytochrome b5 interaction. P450 structure and dynamics were examined with the carbon monoxide (CO) flash photolysis technique. Modulation of the kinetics of CO binding to microsomal P450 by P450 effectors was used to kinetically define individual P450s within the endoplasmic reticulum. This approach showed that the substrate benzphetamine enlarges the CO access channel of P450 2B1 to accelerate CO binding. Polycyclic aromatic hydrocarbons of varying sizes and shapes likewise enhanced CO binding to P450 1A1. The largest rate increases were observed with the smaller tricyclics, while the larger tetracyclics and pentacyclics yielded more modest increases. Flash photolysis studies were also carried out on baculovirus expressed human P450 3A4. At least two P450 conformers were identified which differed in their response to common substrates. The CO binding kinetics is thus a valuable probe of the structure/dynamics of individual P450s in a native membrane environment.